1. Field
This invention relates generally to the genetically engineered mammalian cell lines for the production of biologically active protein. Specifically, the invention is concerned with human hybrid cell clones derived from the fusion process of human embryonic kidney (293S) cells and Burkitt's lymphoma cells. These human hybrid cells can be used for the production of heterologous proteins.
2. Background
To date, most therapeutic recombinant proteins have been produced from non-human mammalian cells. Some examples are:
Chinese hamster ovary (CHO) (dhfr-) cells (Urlaub et al., 1980, Proc Natl Acad Sci U.S.A. 77: 4216-4220) with the amplifiable selection marker dihydrofolate reductase (Kaufman et al., 1982, Mol. Biol. 159: 601-621; Gasser et al., 1982, Proc Natl Acad Sci U.S.A. 79: 6522-6526) have been used for the production of therapeutic recombinant protein.
A variety of recombinant therapeutic proteins are known to be produced in mammalian cells, e.g. recombinant factor VIII (rFVIII) (Kaufman et al., 1988, J Biol Chem 263: 6352-6362), tissue plasminogen activator (tPA) (U.S. Pat. No. 4,766,075 to Goeddel et al., 1988), erythropoietin (EPO) (U.S. Pat. No. 4,703,008 to Lin, 1987), and monoclonal antibodies (mAbs) (U.S. Pat. No. 4,816,397 to Boss et al., 1989).
Baby hamster kidney (BHK) cells (BHK21) were used for production of rFVIII after G418 selection and methotrexate (MTX) amplification of G418 resistant cells (U.S. Pat. No. 4,965,199 to Capon et al., 1990).
Mouse myeloma (NS0) cells were used for production of engineered human anti-TNF antibody (EHAT). (U.S. Pat. No. 4,816,397 to Boss et al., 1989). However, this cell line produces proteins having mouse specific carbohydrate patterns which are not favored for human use.
A human cell line, Namalwa (of Burkitt's lymphoma origin), was used for production of alpha-interferon by Wellcome Research Laboratory and for the production of pro-urokinase (Satoh et al., 1996, Cytotechnology 18: 167-185, 1996), tissue-plasminogen activator (t-PA) (Khan et al., 1995, Biochem Soc Trans 23: S99), granulocyte-macrophage colony-stimulating factor (Okamoto et al., 1991, Arch Biochem Biophys 286: 562-568), interferons and lymphotoxin (Hosoi et al., 1991, Cytotechnology 5: 17-34), and granulocyte colony stimulating factor (Hosoi et al., 1991, Cytotechnology 7: 25-32) by Tokyo Research Laboratories. However, these cells were very difficult to transfect with DNA.
Walls et al. (1989, Gene 81: 139-149) have reported on the use of the dhfr/MTX co-amplification strategy to express functional protein C in human embryonic kidney cells (293S cells). 293 cells (Stillman et al., 1985, Mol. Cell. Biol. 5: 2051-2060) are known as making large aggregates in suspension, especially under high calcium concentration (&gt;100 .mu.M), which promotes larger aggregation and lower cell viability (Peshwa et al., 1993, Biotech and Bioeng 41: 179-187). All references cited are herein incorporated by reference.